Muscle tissue is subdivided into skeletal muscle, cardiac muscle, and smooth muscle tissue and can be considered the biggest organ of a vertebrate. For example, an average adult human male is made up of 40 to 50% skeletal muscle. Skeletal muscle and cardiac muscle belong to the striated muscle tissue and share many functional aspects. For example, the process of excitation-contraction coupling in skeletal muscle cells and cardiac muscle cells (cardiomyocytes) is very similar. Membrane depolarization of the myocytes causes calcium influx via activated voltage-gated L-type calcium channels into the cytoplasm (sarcoplasm) of the myocyte. The rise of the cytoplasmic calcium concentration leads to calcium release from the sarcoplasmic reticulum (SR) by activation of ryanodine receptors (RyR) through the calcium-induced calcium release (CICR) mechanism, and thus, to a further rapid rise of the cytoplasmic calcium concentration. Calcium molecules diffuse through the cytoplasm and bind to contractile proteins such as troponin C which causes contraction of the myocytes. After contraction, calcium is cleared from the cytoplasm by re-uptake of calcium into the sarcoplasmic reticulum mainly by the action of a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). These events are essentially identical in skeletal muscle cells and cardiac muscle cells with minor differences in the isoforms of the involved proteins. For example, while RyR1 is the predominant sarcoplasmic reticulum calcium release channel in skeletal muscle cells, RyR2 is predominant in cardiomyocyte. Similarly, the skeletal muscle sarcoplasmic/endoplasmic reticulum calcium ATPase is SERCA1a, whereas SERCA2a is cardiomyocyte-specific.
Calcium cycling in myocytes is regulated by a plethora of proteins. For example, S100A1 belonging to the S100 protein family (the largest EF-hand calcium-binding protein subfamily) has been reported to interact with both the RyR calcium release channel and SERCA. S100A1 stabilizes RyR in diastole reducing the frequency of calcium sparks and augments calcium release during systole. Furthermore, S100A1 increases SERCA activity during the relaxation phase and it was found to increase contractile function in cardiac muscle as well as skeletal muscle cells. It has been shown that a carboxy-terminal peptide derived from the S100A1 protein mimics the inotropic effect of the full-length S100A1 protein (Most P. et al., 2007, Am. J. Physiol. Regul. Integr. Comp. Physiol. 293:R568-577; Voelkers M. et al., 2007, Cell Calcium 41:135-143).
Defective calcium cycling in myocytes, for example, reduces calcium release from the sarcoplasmic reticulum during contraction, causes aberrant calcium release events, calcium leakage from the sarcoplasmic reticulum, or slowed calcium clearance from the cytoplasm, resulting in a variety of myopathies, i.e., diseases associated with muscular malfunction. For example, cardiac insufficiency, contractile ventricular dysfunction, arrhythmias, heart failure, cardiogenic shock, myocardial infarction, and dysfunction of heart valves have been associated with dysregulation of calcium handling in cardiomyocytes. Analogously, defective calcium cycling in skeletal muscle fibers has been linked with muscular dystrophy (Hopf F. W. et al., 2007, Subcell. Biochem. 45:429-64). Furthermore, mutations in the RyR calcium release channels causing disruption of calcium signaling in muscle cells have been associated with myopathies. In particular, more than 80 mutations have been identified in the skeletal muscle RyR1 calcium release channel and have been linked to malignant hyperthermia, central core disease, or multi-minicore disease. Furthermore, more than 40 mutations in the cardiac RyR2 calcium release channel leading to ventricular arrhythmias and sudden cardiac death have been reported (Dulhunty A. F. et al., 2006, J. Muscle Res. Cell Motil. 27:351-365).
At present, there are no clinical inotropic therapies available for skeletal muscle disorders. Approved therapeutics currently available for the inotropic treatment of cardiomyopathies, such as glycoside derivatives, catecholamines, and phosphodiesterase inhibitors, are afflicted with severe side effects such as increased heart rate and life threatening proarrhythmogenic potential. Besides these approved therapeutics, the S100A1 protein has been suggested as therapeutic in cardiomyopathies, since it was shown that myocardial levels of S100A1 are decreased in heart failure and that S1 00A1 delivery to cardiomyocytes results in an increase of isometric contraction followed by an increase in the amount of calcium pumped into the sarcoplasmic reticulum. However, the administration of S100A1 to a patient with the purpose of treating myopathies requires the delivery route of gene therapy, for example, using viral delivery, with all its well-known side effects and disadvantages (Most P. et al., 2007, Am. J. Physiol. Regul. Integr. Comp. Physiol. 293:R568-577, WO 2008/054713, and Vinge L. E. et al., 2008, Circ. Res. 102:1458-1470).
Therefore, there is an urgent need for novel therapeutics for the inotropic treatment of myopathies, preferably myopathies associated with dysregulation of calcium cycling in muscle cells, which do not exhibit the severe side effects observed for the approved therapeutics and which do not require the high risk delivery route of gene therapy. Regarding skeletal muscle diseases, there is an urgent need for any inotropic therapeutics having the ability to increase the contractile performance of skeletal muscle cells and/or reducing calcium-induced apoptotic cell death in skeletal muscle cells.
A 20 amino acid peptide derived from the calcium binding protein S100 is known to exhibit inotropic effects (Voelkers M. et al., 2007, Cell Calcium 41:135-143). While peptides are considered very useful therapeutic agents, they generally suffer from some drawbacks such as problematic delivery into cells, i.e. cross-membrane transportation, or immunogenic effects. The present inventors surprisingly found that a significantly shortened peptide S100A1Ct6/10 comprising amino acids 76-85 of the S100 protein has inotropic effects similar to said 20-mer peptide. Further fragmentation, however, did not yield functional peptides, suggesting that S100A1ct6/10 comprises the shortest functional S100A1 fragment (PCT/EP 2010/002343). Very surprisingly in view of this, the present inventors now found that peptides comprising even shorter S100A1 fragments also exhibit inotropic effects and they identified a 4 amino acid core motif derived from S100A1 (amino acids 79-82 of the S100A1 protein) which is minimally required for functionality. Due to the reduced size of these shortened peptides, delivery into cells is facilitated and immunogenic side effects are minimized. When administered parenterally the shortened peptides are useful for the treatment of myopathies, such as cardiac and skeletal muscle disorders, without exhibiting mentionable side effects and without requiring gene therapy. For example, the peptides according to the present invention enhance and restore inotropy in normal and failing myocardium as well as in normal and diseased skeletal muscle, enhance and restore sarcoplasmic reticulum calcium handling, prevent calcium induced apoptotic cell death in myocytes, protect from proarrhythmogenic sarcoplasmic reticulum calcium leak and tachyarrhythmias, and prevent cardiac death due to protection from pump failure and tachyarrhythmias. The peptides of the present invention are particularly useful for enhancing contractile performance of cardiac and skeletal muscle tissue without major side effects.